The quantification of intercellular variability and biological noise is a fundamental aspect of the characterization of synthetic gene circuits [1]. Since the most common techniques used for this evaluation (flow cytometers and complex microscopy set-ups) are expensive and generally unaffordable by small laboratories, we have developed MUSIQ (Microscope flUorescence SIngle cell Quantification), a protocol that combines a standard fluorescence microscope set-up with a custom made software and allows the quantitative evaluation of the fluorescent signal emitted by single E. coli cells [2]. MUSIQ was entirely validated, through a comparative analysis with standard cytofluorimetric assays. These results, here fully reported, determined the equivalence of the two methods on a wide range of fluorescence intensities, both in terms of average signal and phenotypic variability. While our approach was determined to have a slightly lower sensitivity, with respect to the flow cytometer, it allows for a more flexible analysis that can include the study of cellular morphology and culture spatial patterns. Furthermore it is accessible to a large base of researchers, relying on general purpose instrumentation and freeware software  [3]. These characteristics determine MUSIQ to be an affordable alternative for the evaluation of intercellular variability in bacterial cultures and thus a significant advancement in synthetic biology, that will improve the accuracy of gene expression quantification and the functional characterization of synthetic gene circuits. [1] Bandiera et al. Front Microbiol. 2016. doi:10.3389/fmicb.2016.00479. [2] Cortesi et al. Journal of Biological Engineering (2017) 11:8 [3] http://www.mcbeng.it/en/downloads/software/musiq.html


MUSIQ: a reliable method for the quantification of E. coli single cell fluorescence distributions using a standard microscope set-up

Marilisa Cortesi;L. Bandiera;A. Pasini;A. Bevilacqua;A. Gherardi;S. Furini;E. Giordano
2017

Abstract

The quantification of intercellular variability and biological noise is a fundamental aspect of the characterization of synthetic gene circuits [1]. Since the most common techniques used for this evaluation (flow cytometers and complex microscopy set-ups) are expensive and generally unaffordable by small laboratories, we have developed MUSIQ (Microscope flUorescence SIngle cell Quantification), a protocol that combines a standard fluorescence microscope set-up with a custom made software and allows the quantitative evaluation of the fluorescent signal emitted by single E. coli cells [2]. MUSIQ was entirely validated, through a comparative analysis with standard cytofluorimetric assays. These results, here fully reported, determined the equivalence of the two methods on a wide range of fluorescence intensities, both in terms of average signal and phenotypic variability. While our approach was determined to have a slightly lower sensitivity, with respect to the flow cytometer, it allows for a more flexible analysis that can include the study of cellular morphology and culture spatial patterns. Furthermore it is accessible to a large base of researchers, relying on general purpose instrumentation and freeware software  [3]. These characteristics determine MUSIQ to be an affordable alternative for the evaluation of intercellular variability in bacterial cultures and thus a significant advancement in synthetic biology, that will improve the accuracy of gene expression quantification and the functional characterization of synthetic gene circuits. [1] Bandiera et al. Front Microbiol. 2016. doi:10.3389/fmicb.2016.00479. [2] Cortesi et al. Journal of Biological Engineering (2017) 11:8 [3] http://www.mcbeng.it/en/downloads/software/musiq.html

Atti del Congresso Biennale dell' Associazione Nazionale Biologia Cellulare e Differenziamento (ABCD)
Marilisa, Cortesi; Bandiera, L.; Pasini, A.; Bevilacqua, A.; Gherardi, A.; Furini, S.; Giordano, E.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/626155
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